The proper localization and dynamics of signaling events are essential for achieving the efficiency, specificity and diversity of intracellular signaling. We plan to study how signals are transmitted to the actin cytoskeleton in cells to dictate local assembly and remodeling in response to tyrosine kinase mediated signaling and following adhesion to extracellular matrix (ECM) proteins. To aid in these studies, we also propose to develop genetically encoded FRET-based kinase activity reporters (biosensors) to study the spatiotemporal patterns of protein kinase activity within single living cells. Specifically, we plan to investigate the pathways used by the c-Abl tyrosine kinase to regulate assembly of the actin cytoskeleton and promote formation of surface filopodia in cells spreading on ECM. We will test the model that the Dokl adaptor is phosphorylated by c-Abl in fibroblasts spreading on fibronectin, and that this leads to recruitment of the Nck SH2/3 adaptor protein. We will study how Nck transmits a Rho family-independent signal to initiate F-actin assembly. We will determine precisely where and when this pathway is activated in spreading cells, using Abl kinase biosensors and GFP-fusion proteins, to establish how this elicits formation of F-actin containing filopodia. In addition, we will analyze the EphA2 receptor tyrosine kinase signaling pathways that are activated in fibroblasts spreading on an ephrin A1 ligand coated surface, and lead to activation of integrins and assembly of the actin cytoskeleton, focusing on the mechanisms of activation of Rap 1a and Rac by p 130Cas/Crk complexes, signaling by Src/FAK complexes, and the coupling of Rac/Cdc42 activation to the characteristic reorganization of the F-actin cytoskeleton observed in spreading fibroblasts. We will determine where and when these pathways are activated in spreading cells using biosensors and GFP-fusion proteins, and apply what we learn to ephrin/Eph signaling in neuronal cells. Finally, we propose to develop multiple FRET-based biosensor proteins that specifically report the activities of members of the ERK, JNK and p38 families of MAP kinases, and of ATM/ATR PIKK family kinases involved in the DNA damage response, and employ these reporters for studies in living cells in culture and during C. elegans and zebrafish development to determine where and when these protein kinases are activated. The overall goal is to investigate at high resolution the spatial and temporal dynamics of protein phosphorylation-mediated signal transduction events within the cell, and define how this generates localized outputs in the cell, such as the organization of the actin cytoskeleton, in response to external stimuli.